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Background: Homologous Recombination (HR) is the most accurate repair pathway for double-strand breaks and replication fork disruption that is capable of faithfully restoring the original nucleotide sequence of the broken DNA. The deficiency in this mechanism is a frequent event in tumorigenesis. Therapies that exploit defects in HR have been explored essentially in breast, ovarian, pancreas, and prostate cancers, but poorly in colorectal cancers (CRC) although CRC ranks second in mortality worldwide. Methods: Tumor specimens and matched healthy tissues from 63 patients with CRC were assessed for gene expression of HR key components and MMR status, which were correlated with clinicopathological features, progression-free survival, and overall survival (OS). Results: Enhanced expression of MRE11A, the gene encoding a key molecular actor for resection is significantly overexpressed in CRC, is associated with the occurrence of primary tumors particularly T3-T4 and is found in more than 90% of the right-side of CRC, the location with worst prognostic. Importantly, we also found that high MRE11A transcript abundance is associated with 16.7 months shorter OS and a 3.5 higher risk of death. Conclusion: Monitoring MRE11 expression could be used both as a predictor of outcome and as a marker to select CRC patients for treatments thus far adapted for HR deficient cancers.

Background The advances in colorectal cancer (CRC) treatment include the identification of deficiencies in Mismatch Repair (MMR) pathway to predict the benefit of adjuvant 5-fluorouracil (5-FU) and oxaliplatin for stage II CRC and immunotherapy. Defective MMR contributes to chemoresistance in CRC. A growing body of evidence supports the role of Poly-(ADP-ribose) polymerase (PARP) inhibitors, such as Olaparib, in the treatment of different subsets of cancer beyond the tumors with homologous recombination deficiencies. In this work we evaluated the effect of Olaparib on 5-FU cytotoxicity in MMR-deficient and proficient CRC cells and the mechanisms involved. Methods Human colon cancer cell lines, proficient (HT29) and deficient (HCT116) in MMR, were treated with 5-FU and Olaparib. Cytotoxicity was assessed by MTT and clonogenic assays, apoptosis induction and cell cycle progression by flow cytometry, DNA damage by comet assay. Adhesion and transwell migration assays were also performed. Results Our results showed enhancement of the 5-FU citotoxicity by Olaparib in MMR-deficient HCT116 colon cancer cells. Moreover, the combined treatment with Olaparib and 5-FU induced G2/M arrest, apoptosis and polyploidy in these cells. In MMR proficient HT29 cells, the Olaparib alone reduced clonogenic survival, induced DNA damage accumulation and decreased the adhesion and migration capacities. Conclusion Our results suggest benefits of Olaparib inclusion in CRC treatment, as combination with 5-FU for MMR deficient CRC and as monotherapy for MMR proficient CRC. Thus, combined therapy with Olaparib could be a strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC.

Background: Snake venoms are natural sources of proteins and peptides with several biological activities. Therefore, this study aimed to evaluate the in vitro toxicity of crude Bothrops jararaca (B. jararaca) venom on different human tumor cells and investigate its action mechanism. Material and methods: The crude venom from B. jararaca was provided by State Foundation for Health Research and Production and Center of Toxicological Information of Rio Grande do Sul, Brazil. To investigate different sensitivities of normal and cancer cell lines, cell viability was measured by using the MTT and neutral red uptake assays.

Resumo Fundamento A doxorrubicina (DOX) é frequentemente usada para tratar muitos tipos de cânceres, apesar da cardiotoxicidade dose-dependente. Como alternativa, o resveratrol é um polifenol que tem demonstrado efeitos cardioprotetores em vários modelos de disfunção cardíaca. Objetivo Este estudo investigou se o tratamento com resveratrol em ratas gestantes protege contra toxicidade induzida por doxorrubicina em cardiomiócitos da ninhada. Métodos Ratas Wistar (n-8) receberam sresveratrol como suplemento alimentar durante a gestação. No nascimento da ninhada, os corações (9-11) foram usados para se obter a cultura primária de cardiomiócitos. A cardiotoxicidade induzida por DOX e os efeitos da suplementação com resveratrol foram avaliados por marcadores de stress oxidativo, tais como oxidação da diclorofluoresceína diacetato, diminuição da atividade de enzimas antioxidantes, e oxidação do teor total de grupos sulfidrila, além da avaliação da viabilidade celular, geração de danos ao DNA, bem como a resposta de reparo aos danos ao DNA. Um valor de p

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians’ skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions ofR. marinasecretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active againstStaphylococcus aureus,Pseudomonas aeruginosa,Escherichia coli,Plasmodium falciparum,Leishmania guyanensis, andLeishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents ofR.marinasecretion as well as the bioactive potential of these molecules.

Mucopolysaccharidosis type II (MPS II or Hunter syndrome) is an inborn error of metabolism characterized by the accumulation of glycosaminoglycans (GAG) in lysosomes. Enzyme replacement therapy (ERT) can reduce GAG storage, ameliorate symptoms, and slow disease progression. Oxidative damages may contribute to the MPS II pathophysiology, and treatment with ERT might reduce the effects of oxidative stress. We evaluated levels of DNA damage (including oxidative damage) and chromosome damage in leukocytes of long-term-treated MPS II patients, by applying the buccal micronucleus cytome assay. We observed that, despite long-term ERT, MPS II patients had higher levels of DNA damage and higher frequencies of micronuclei and nuclear buds than did control. These genetic damages are presumably due to oxidation: we also observed increased levels of oxidized guanine species in MPS II patients. Therapy adjuvant to ERT should be considered, in order to decrease oxidative damage and cytogenetic alterations.

Cocaine is one of the most popular illicit drug worldwide. Due its great addictive potential, which leads to euphoria and hyperactivity, it is considered a public health concern. At the central nervous system, the drug acts inhibiting catecholamine re-uptake. It is now known that in addition to the toxicity of the drug itself, the contaminants present in the street drug have raised concern about the harmful effects on health. Toxicological in vivo and in vitro studies have demonstrated the toxic effects of cocaine correlated with the generation of reactive oxygen species (ROS), which in turn lead to oxidative damage to the cells. Therefore the aim of this work was to propose an in vitro model that reunites the main parameters of toxicity of the cocaine already observed in the literature so far, and we tested this model using cocaine and seizure cocaine sample (SCS), kindly provided by Federal Police of Brazil. For that, we used a C6 glioblastoma cells and evaluated cell death, oxygen reactive species induction, oxidation of macromolecules as membrane lipids and DNA and loss of mitochondrial membrane potential after cocaine exposure. The results showed that cocaine can decrease cellular viability in a dose-dependent way in the C6 cell immortalized and astrocytes primary culture. Cocaine also induced cellular death by apoptosis. However, in the seizure cocaine sample (SCS), the predominant cell death was due to necrosis. Using dichlorofluorescein (DCF) assay, we confirmed ROS production after cocaine exposition. In agreement with these findings, occurred an increasing in MDA production, as well as increased superoxide dismutase (SOD) and catalase (CAT) activity. The induction of DNA damage was observed after cocaine. Our results demonstrate the occurrence of mitochondrial dysfunction by depolarization of mitochondrial membrane as a consequence of cocaine treatment. In summary, these results demonstrated that cocaine can induce reactive oxygen species formation, leading to oxidative stress. As a consequence of this unbalance, DNA damage, lipidic peroxidation and loss of mitochondrial membrane occurred, which could be an answer to cell death observed.

Introduction: Homocysteine (Hcy) tissue accumulation occurs in a metabolic disease characterized biochemically by cystathionine β-synthase (CBS) deficiency and clinically by mental retardation, vascular problems, and skeletal abnormalities. Previous studies indicate the occurrence of DNA damage secondary to hyperhomocysteinemia and it was observed that DNA damage occurs in leukocytes from CBS-deficient patients. This study aimed to investigate whether an oxidative mechanism could be involved in DNA damage previously found and investigated the in vitro effect of N-acety-L-cysteine (NAC) on DNA damage caused by high Hcy levels. Methods: We evaluated a biomarker of oxidative DNA damage in the urine of CBS‑deficient patients, as well as the in vitro effect of NAC on DNA damage caused by high levels of Hcy. Moreover, a biomarker of lipid oxidative damage was also measured in urine of CBS deficient patients. Results: There was an increase in parameters of DNA (8-oxo-7,8-dihydro-2’- deoxyguanosine) and lipid (15-F2t-isoprostanes levels) oxidative damage in CBS-deficient patients when compared to controls. In addition, a significant positive correlation was found between 15-F2t-isoprostanes levels and total Hcy concentrations. Besides, an in vitro protective effect of NAC at concentrations of 1 and 5 mM was observed on DNA damage caused by Hcy 50 μM and 200 μM. Additionally, we showed a decrease in sulfhydryl content in plasma from CBS-deficient patients when compared to controls. Discussion: These results demonstrated that DNA damage occurs by an oxidative mechanism in CBS deficiency together with lipid oxidative damage, highlighting the NAC beneficial action upon DNA oxidative process, contributing with a new treatment perspective of the patients affected by classic homocystinuria. Keywords: Cystathionine-β-synthase deficiency; oxidative stress; 8-oxo-7,8-dihydro- 2’-deoxyguanosine; homocysteine; DNA damage; N-acetyl-L-cysteine

Abstract Aim: To evaluate the prognostic value of molecular modulation of double strand break (DSBR) - XRCC2 and XRCC5 - core components; DNA damage tolerance/translesion synthesis (DDT/TLS) - POLH, POLK and POLQ – and, finally, of interstrand crosslink repair (ICLR) -DCLRE1A – pathways in sporadic colorectal cancer (CRC) progression. Method: Tumour specimens and matched health mucosal tissues from 47 patients with CRC who underwent surgery were assessed for gene expression of XRCC2, XRCC5, POLH, POLK, POLQ and DCLRE1A by qRT-PCR; protein expression of Polk, Ku80, p53, Ki67 and mismatch repair MLH1 and MSH2 components were assessed by immunohistochemistry (IHC); CpG island promoter methylation of XRCC5, POLH, POLK, POLQ and DCLRE1A was performed. Uni and multivariate analyses were employed to determine associations with clinicopathological features and prognostic value of molecular data. Results: Neoplastic tissues exhibited induction of POLK (p

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of R. marina secretion were also evaluated. Fractionation prior to filtration from poison showed separation of low mass content (steroids and alkaloids) and high molecular mass (protein). Material below 10 kDa two steroids, marinobufagin and desacetylcinobufagin, was detected. Crude extract and fractions were active against Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, Plasmodium falciparum, Leishmania guyanensis, and Leishmania braziliensis. Crude extract was also active against cancer cells although it was not cytotoxic for normal cells. This extract did not show significant DNA damage but it showed an important inflammatory effect in vivo. The information obtained in this work contributes to the understanding of the constituents of R. marina secretion as well as the bioactive potential of these molecules.

Skin secretions of frogs have a high chemical complexity. They have diverse types of biomolecules, such as proteins, peptides, biogenic amines, and alkaloids. These compounds protect amphibians' skin against growth of bacteria, fungi, and protozoa and participate in defense system against attack from predators. Therewith, this work performed biochemical and biological profile of macroglands parotoid secretion from cane toad. For poison analysis, we performed molecular exclusion and reverse phase chromatography, electrophoresis, and mass spectrometry. Antimicrobial, antiplasmodial, leishmanicidal, cytotoxicity, genotoxicity, and inflammatory activity of crude and/or fractions of

Combretum leprosumMart., a member of the Combretaceae family, is a traditionally used Brazilian medicinal plant, although no evidence in the literature substantiates its antioxidant action and the safety of its use. We evaluated the antioxidant properties of the ethanolic extract (EE) from flowers ofC. leprosumand its isolated products 5,3′-dihydroxy-3,7,4′-trimethoxyflavone (FCL2) and 5,3′,4′-trihydroxy-3,7-dimethoxyflavone (FCL5) inSaccharomyces cerevisiaestrains proficient and deficient in antioxidant defenses. Their mutagenic activity was also assayed inS. cerevisiae,whereas cytotoxic and genotoxic properties were evaluated by MTT and Comet Assays, respectively, in V79 cells. We show that the EE, FCL2, and FCL5 have a significant protective effect against H2O2. FCL2 showed a better antioxidant action, which can be related to the activation of the 3′-OH in the presence of a methoxyl group at 4′ position in the B-ring of the molecule, while flavonoids did not induce mutagenesis in yeast, and the EE was mutagenic at high concentrations. The toxicity of these compounds in V79 cells increases from FCL2 = FCL5 < EE; although not cytotoxic, FCL5 induced an increase in DNA damage. The antioxidant effect, along with the lower toxicity and the absence of genotoxicity, suggests that FCL2 could be suitable for pharmacological use.

The present study evaluates antigenotoxic and antimutagenic properties of diphenyl ditelluride (DPDT) against several known mutagens in Chinese hamster lung fibroblasts (V79 cells). DPDT was not cytotoxic and genotoxic at concentrations ranging from 0.01 to 0.1 μM. The pre-treatment for 2h with this organotellurium compound at non-cytotoxic dose range (0.01, 0.05 and 0.1 μM) increased cell survival after challenge with hydrogen peroxide (H2O2), t-butyl hydroperoxide (t-BOOH), methylmethanesulphonate (MMS) or ultraviolet (UV)C radiation. In addition, the pre-treatment with DPDT decreased the DNA damage and Formamidopyrimidine DNA-glycosylase (Fpg)- and Endonuclease III (Endo III) sensitive sites induction by the studied genotoxic agents, as verified by comet assay and modified comet assay, respectively. The pre-treatment also reduced micronucleus frequency, revealing the protector effect of DPDT against MMS and UVC-induced mutagenesis. Our results demonstrate that DPDT-treated cells at concentration range of 0.01-0.1 μM do not change thiobarbituric acid reactive species (TBARS) levels and ROS generation. Moreover, DPDT pre-treatment at this concentration range decreases the ROS induction by H2O2 and t-BOOH treatment indicating antioxidant potential. On the other hand, concentrations higher than 0.1 μM increase TBARS formation and inhibited superoxide dismutase (SOD) activity, suggesting pro-oxidative effect of this compound at high concentrations. Our results suggest that DPDT presents antigenotoxic and antimutagenic properties at concentration range of 0.01-0.1 μM. The protection effect could be attributed to antioxidant capacity of DPDT at this concentration range in V79 cells.

Mucopolysaccharidosis type IVA (MPS IVA) is an inborn error of glycosaminoglycan (GAG) catabolism due to the deficient activity of N-acetylgalactosamine-6-sulfate sulfatase that leads to accumulation of the keratan sulfate and chondroitin 6-sulfate in body fluids and in lysosomes. The pathophysiology of this lysosomal storage disorder is not completely understood. The aim of this study was to investigate oxidative stress parameters, pro-inflammatory cytokine and GAG levels in MPS IVA patients. We analyzed urine and blood samples from patients under ERT ( n = 17) and healthy age-matched controls ( n = 10–15). Patients presented a reduction of antioxidant defense levels, assessed by a decrease in glutathione content and by an increase in superoxide dismutase activity in erythrocytes. Concerning lipid and protein damage, it was verified increased urine isoprostanes and di-tyrosine levels and decreased plasma sulfhydryl groups in MPS IVA patients compared to controls. MPS IVA patients showed higher DNA damage than control group and this damage had an oxidative origin in both pyrimidine and purine bases. Interleukin 6 was increased in patients and presented an inverse correlation with GSH levels, showing a possible link between inflammation and oxidative stress in MPS IVA disease. The data presented suggest that pro-inflammatory and pro-oxidant states occur in MPS IVA patients even under ERT. Taking these results into account, supplementation of antioxidants in combination with ERT can be a tentative therapeutic approach with the purpose of improving the patient's quality of life. To the best of our knowledge, this is the first study relating MPS IVA patients with oxidative stress.

Colorectal cancer (CRC) is the third major cause of cancer-related deaths worldwide. However, despite the scientific efforts to provide a molecular classification to improve CRC clinical practice management, prognosis and therapeutic decision are still strongly dependent on the TNM staging system. Mismatch repair system deficiencies can occur in many organs, but it is mainly a hallmark of CRC influencing clinical outcomes and response to therapy. This review will discuss the effect of the modulation of other DNA repair pathways (direct, excision and double strand break repairs) in the clinical and pathological aspects of colorectal cancer and its potential as prognostic and predictive biomarkers.

// Natalia M. Leguisamo 1 , Helena C. Gloria 1 , Antonio N. Kalil 1, 2 , Talita V. Martins 2 , Daniel B. Azambuja 1, 2 , Lisiane B. Meira 3, * and Jenifer Saffi 1, * 1 Genetic Toxicology, Universidade Federal de Ciencias da Saude de Porto Alegre (UFCSPA), Porto Alegre, Rio Grande do Sul, Brazil 2 Oncology and Colorectal Surgery, Santa Casa de Misericordia de Porto Alegre (ISCMPA), Porto Alegre, Rio Grande do Sul, Brazil 3 Department of Clinical and Experimental Medicine, Faculty of Health and Medical Sciences, University of Surrey, Guildford, United Kingdom * These authors share senior authorship Correspondence to: Natalia M. Leguisamo, email: nmleguisamo@gmail.com Keywords: colorectal cancer, base excision repair, prognosis, energy metabolism, temozolomide Received: February 29, 2016 Accepted: November 30, 2016 Published: January 31, 2017 ABSTRACT Colorectal cancer (CRC) is prevalent worldwide, and treatment often involves surgery and genotoxic chemotherapy. DNA repair mechanisms, such as base excision repair (BER) and mismatch repair (MMR), may not only influence tumour characteristics and prognosis but also dictate chemotherapy response. Defective MMR contributes to chemoresistance in colorectal cancer. Moreover, BER affects cellular survival by repairing genotoxic base damage in a process that itself can disrupt metabolism. In this study, we characterized BER and MMR gene expression in colorectal tumours and the association between this repair profile with patients’ clinical and pathological features. In addition, we exploited the possible mechanisms underlying the association between altered DNA repair, metabolism and response to chemotherapy. Seventy pairs of sporadic colorectal tumour samples and adjacent non-tumour mucosal specimens were assessed for BER and MMR gene and protein expression and their association with pathological and clinical features. MMR-deficient colon cancer cells (HCT116) transiently overexpressing MPG or XRCC1 were treated with 5-FU or TMZ and evaluated for viability and metabolic intermediate levels. Increase in BER gene and protein expression is associated with more aggressive tumour features and poor pathological outcomes in CRC. However, tumours with reduced MMR gene expression also displayed low MPG , OGG1 and PARP1 expression. Imbalancing BER by overexpression of MPG , but not XRCC1 , sensitises MMR-deficient colon cancer cells to 5-FU and TMZ and leads to ATP depletion and lactate accumulation. MPG overexpression alters DNA repair and metabolism and is a potential strategy to overcome 5-FU chemotherapeutic resistance in MMR-deficient CRC.

Chromatin is thought to modulate access of repair proteins to DNA lesions, and may be altered by chromatin remodelers to facilitate repair. We investigated the participation of chromatin remodelers and DNA repair in 5-fluorouracil (5-FU) cytotoxicity in Saccharomyces cerevisiae. 5-FU is an antineoplastic drug commonly used in clinical settings. Among the several strains tested, only those with deficiencies in ATP-dependent chromatin remodeling (CR) and some histone acetyltransferases (HAT) exhibited sensitivity to 5-FU. CR and HAT double-mutants exhibited increased resistance to 5-FU in comparison to the wild-type mutant, but were still arrested in G2/M, as were the sensitive strains. The participation of Htz1p in 5-FU toxicity was also evaluated in single- and double-mutants of CR and HAT; the most significant effect was on cell cycle distribution. 5-FU lesions are repaired by different DNA repair machineries, including homologous recombination (HR) and post-replication repair (PRR). We investigated the role of CR and HAT in these DNA repair pathways. Deficiencies in Nhp10 and CR combined with deficiencies in HR or PRR increased 5-FU sensitivity; however, combined deficiencies of HAT, HR, and PRR did not. CRs are directly recruited to DNA damage and lead to chromatin relaxation, which facilitates access of HR and PRR proteins to 5-FU lesions. Combined deficiencies in HAT with defects in HR and PRR did not potentiate 5-FU cytotoxicity, possibly because they function in a common pathway.

Platonia insignis Mart., a native species of the Brazilian Amazon more commonly known as bacuri, is a member of the Clusiaceae family. In this study, we evaluated the chemical composition and the antioxidant and toxicity activities of the dichloromethane and ethyl acetate fractions from P. insignis seed ethanolic extract using different experimental models. Our results demonstrate in vitro antioxidant effects, by 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt and 1,1-diphenyl-2-picryl-hydrazyl assays, as well as in vivo effects in antioxidant-defective Saccharomyces cerevisiae strains to both fractions. Toxicity was evaluated against the micro-crustaceous Artemia salina Leach. and promastigote Leishmania amazonensis. The dichloromethane fraction was the most active fraction evaluated on A. salina and promastigote L. amazonensis (IC(50) = 24.89 μg/mL and 2.84 μg/mL, respectively). In addition, a slight cytotoxicity was observed in mammalian V79 cells using ethyl acetate and dichloromethane fractions with MTT assays. Both fractions displayed genotoxicity up to 25 μg/mL (dichloromethane) and 10 μg/mL (ethyl acetate) in V79 cells, as evaluated by the alkaline comet assay. Thus, in this study, we demonstrate for the first time that ethyl acetate and dichloromethane fractions from P. insignis seeds display antioxidant effects, a toxic effect against A. salina and L. amazonensis and induce genotoxicity in V79 mammalian cells. The observed activities can be attributed to the phenolic compounds present in these fractions and to the presence of xanthones (alpha- and gamma-mangostin).

The essential oil (EO) of aerial parts of Lantana camara L., Verbenaceae, from Simões, Piaui, Northeast of Brazil, were obtained by hydrodistillation and analyzed by GC-FID and GC-MS techniques. In total, 68 compounds were identified. The most representative compounds of the oil were mono and sesquiterpenes. The main compounds found in the oil of the leaves in different months were β-caryophyllene (10.5%, in June of 2009), sabinene (7.98%, in September of 2008), limonene (7.68%, in September of 2008), spathulenol (11.64%, in September of 2008). The oil from stems of L. camara was characterized by a largest amount of sesquiterpenoids, with spatulenol (15.9%) and caryophyllene oxide (17.1% in June of 2009), as main components. β-Gurjunene (32.7%, in September of 2008) was the most prominent compound in the stems oils, which was absent or at very low relative abundance in leaves. L. camara essential oils from leaves were cytotoxic to V79 mammalian cells and also to Artemia salina, showing 50% lethal concentration (LC50) values from 0.23 µg/mL. The in vitro data obtained in this study suggested that EO may also be effective treating yeast infection in patients infected with fluconazole and terbinafine resistant isolates, but its toxicity must be monitored carefully.

Tin or stannous (Sn 2+ ) compounds are used as catalysts, stabilizers in plastic industries, wood preservatives, agricultural biocides and nuclear medicine. In order to verify the Sn 2+ up-take and toxicity in yeast cells we utilized a multi-elemental analysis known as particle-induced X-ray emission (PIXE) along with cell survival assays and quantitative real-time PCR. The detection of Sn 2+ by PIXE was possible only in yeast cells in stationary phase of growth (STAT cells) that survive at 25 mM Sn 2+ concentration. Yeast cells in exponential phase of growth (LOG cells) tolerate only micro-molar Sn 2+ concentrations that result in intracellular concentration below of the method detection limit. Our PIXE analysis showed that STAT XV185-14c yeast cells demonstrate a significant loss of intracellular elements such as Mg, Zn, S, Fe and an increase in P levels after 1 h exposure to SnCl 2 . The survival assay showed enhanced tolerance of LOG yeast cells lacking the low-affinity iron and zinc transporters to stannous treatment, suggesting the possible involvement in Sn 2+ uptake. Moreover, our qRT-PCR data showed that Sn 2+ treatment could generate reactive oxygen species as it induces activation of many stress-response genes, including SOD1 , YAP1 , and APN1 .

Doxorubicin (DOX) is an important tumor chemotherapeutic agent, acting mainly by genotoxic action. This work focus on cell processes that help cell survival, after DOX-induced DNA damage. In fact, cells deficient for XPA or DNA polymerase eta (pol eta, XPV) proteins (involved in distinct DNA repair pathways) are highly DOX-sensitive. Moreover, LY294002, an inhibitor of PIKK kinases, showed a synergistic killing effect in cells deficient in these proteins, with a strong induction of G2/M cell cycle arrest. Taken together, these results indicate that XPA and pol eta proteins participate in cell resistance to DOX-treatment, and kinase inhibitors can selectively enhance its killing effects, probably reducing the cell ability to recover from breaks induced in DNA.

Platonia insignis Mart. (Clusiaceae), commonly known as "bacuri," is a timber and fruit native species of the Brazilian Amazon. Some plants of the Clusiaceae family have their pharmacological properties associated with the presence of xanthone and polycyclic polyprenylated acylphloroglucinols derivatives, which have antioxidant and anticarcinogenic activities.The aim of this study was to assess the in vivo potential of extracts, fractions, and garcinielliptone FC isolated from of Platonia insignis seeds as a natural antioxidant.Male Wistar rats (250-280 g; 2 months old) were treated with Tween 80 0.05% dissolved in 0.9% saline (i.p, vehicle - control group), ethanol extract (EE), hexane extract (HE), dichloromethane fraction (DMF), ethyl acetate fraction (EAF), and garcinielliptone FC (GFC) isolated from P. insignis at doses 2 mg/kg (i.p.). All groups were observed for 24 h after the treatment. The antioxidant enzymatic activities [superoxide dismutase (SOD) and catalase (CAT)] were measured using spectrophotometric methods.There were no marked alterations in SOD and CAT activities in rat hippocampus after pretreatment with EE, HE, DMF, EAF, and GFC. However, the pretreatment with GFC induced a significantly increase of 13, 17, 19, and 13% in SOD activities when compared to EE, HE, DMF, or EAF groups, respectively.Our findings strongly support the hypothesis that GFC isolated from P. insignis has a significant potential to be used as a natural antioxidant agent probably due to the modulation of enzymatic activity of hippocampal SOD.

Introdução: O processo de cicatrização é uma sequência de reações para reconstituir o tecido. O bacurizeiro (Platonia insignis Mart.) é nativo da Amazônia e é usado como cicatrizante e antibiótico. Objetivo: Analisar a cicatrização de feridas cutâneas tratadas com a banha de bacuri. Metodologia: Utilizaram-se 46 ratos Wistar, machos. Confeccionou-se uma ferida de 2 cm de diâmetro no dorso dos animais e em seguida foram distribuídos em dois grupos: controle e tratado com banha de bacuri. Foram avaliados no 4º, 7º e 14º dias do pós-operatório. Resultados: A inflamação aguda reduziu a partir do sétimo dia nos dois grupos, sendo mais evidente no grupo experimental. No quarto dia, ambos os grupos não mostraram reepitelização, já no sétimo, os animais tratados com a banha demonstraram reepitelização considerável. Conclusão: A banha de bacuri foi eficiente no sétimo dia de tratamento. Os ácidos graxos presentes nessa substância favorecem o processo cicatricial.

Twenty pharmacists and nurses handling anti-neoplastic drugs in a hospital were monitored during a working week, from Monday to Friday, in the morning (only on Monday) and afternoon (all days). Genotoxicity was analysed by the comet assay and the micronucleus (MN) test, while oxidative stress was analysed in serum by thiobarbituric acid reactive substances (TBARS) and by measurements of the antioxidant enzymes superoxide dismutase (Sod) and catalase (Cat). The exposed workers presented increased DNA damage levels by the comet assay as compared to the controls. The comet assay results have also shown significant positive correlation with the day of the week and with alcohol consumption. MN frequency was significantly higher in the exposed workers and presented noteworthy correlation with age and working time. In the oxidative stress parameters, only Cat presented a significant increase in the exposed group, considering all the samplings. However, TBARS data showed interesting results, considering the different sampling times; the exposed group presented a significant correlation with the working days and significantly higher results on Friday as compared to the controls and Monday morning. Monitoring occupational risk during a longer time, e.g. during a working week as done in this study, introduces additional aspects of risk behaviour, which can improve risk management. This study demonstrates the usefulness of evaluating oxidative stress also in genotoxic risk assessment since both events often result from the same factors.

The beta-carboline alkaloids found in medical plants and in a variety of foods, beverages and cigarette smoke have a range of action in various biological systems. In vitro studies have demonstrated that these alkaloids can act as scavengers of reactive oxygen species. In this paper, we report the in vivo antioxidative properties of the aromatic (harmane, harmine, harmol) and dihydro-beta-carbolines (harmaline and harmalol) studied by using Saccharomyces cerevisiae strains proficient and deficient in antioxidant defenses. Their antimutagenic activity was also assayed in S. cerevisiae and the antigenotoxicity was tested by the comet assay in V79 cell line, when both eukaryotic systems were exposed to H(2)O(2). We show that the alkaloids have a significant protective effect against H(2)O(2) and paraquat oxidative agents in yeast cells, and that their ability to scavenge hydroxyl radicals contributes to their antimutagenic and antigenotoxic effects.

Background Renal failure is a frequent and serious complication in patients with decompensated cirrhosis. Objectives We aimed to evaluate the renal oxidative stress, cell damage and impaired cell function in animal model of cirrhosis. Methods Secondary biliary cirrhosis was induced in rats by ligation of the common bile duct. We measured TBARS, ROS and mitochondrial membrane potential in kidney as markers of oxidative stress, and activities of the antioxidant enzymes. Relative cell viability was determined by trypan blue dye-exclusion assay. Annexin V-PE was used with a vital dye, 7-AAD, to distinguish apoptotic from necrotic cells and comet assay was used for determined DNA integrity in single cells. Results In bile duct ligation animals there was significant increase in the kidney lipoperoxidation and an increase of the level of intracellular ROS. There was too an increase in the activity of all antioxidant enzymes evaluated in the kidney. The percentage viability was above 90% in the control group and in bile duct ligation was 64.66% and the dominant cell death type was apoptosis. DNA damage was observed in the bile duct ligation. There was a decreased in the mitochondrial membrane potential from 71.40% ± 6.35% to 34.48% ± 11.40% in bile duct ligation. Conclusions These results indicate that intracellular increase of ROS cause damage in the DNA and apoptosis getting worse the renal function in cirrhosis. Contexto A falência renal é uma complicação grave e frequente em pacientes com cirrose descompensada. Objetivo Avaliar o estresse oxidativo, o dano ao DNA e alterações na função celular no rim em um modelo animal de cirrose. Métodos A cirrose biliar secundária foi induzida em ratos através da ligadura do duto biliar comum. Foi medido no rim o TBARS (substâncias que reagem ao ácido tiobarbitúrico), ERO (espécies reativas de oxigênio), o potencial de membrana mitocondrial e a atividade das enzimas antioxidantes. A viabilidade celular foi determinada utilizando o ensaio de exclusão do trypan-blue. Para distinguir células em apoptose ou necrose foram usados os marcadores: Anexina V-PE e 7-AAD e o ensaio cometa foi utilizado para determinar dano ao DNA. Resultados Em animais cirróticos houve um aumento significativo da lipoperoxidação no rim e na quantidade de ERO intracelular. Foi observado um aumento na atividade de todas as enzimas antioxidantes. A porcentagem de viabilidade celular foi superior a 90% no grupo controle e de 64,66% no grupo da ligadura do duto biliar. O padrão de morte celular predominante foi apoptose e houve dano ao DNA no grupo da ligadura do duto biliar. Observou-se uma redução no potencial de membrana mitocondrial no grupo da ligadura do duto biliar (34,48% ± 11,40%) em comparação aos controles (71,40% ± 6,35%). Conclusão Esses resultados parecem indicar que nos animais cirróticos ocorre um aumento no dano oxidativo e ao DNA levando as células renais à apoptose, o que contribui para a falência renal na cirrose.

▼The raw quality of data is one of the first and most desirable requirements of Good Laboratory Practice (GLP) and Good Statistics Practice (GSP). In quantitative microbiology, this implies that cell and colony counts should follow the ‘ideal’ Poisson distribution (Ref. 1). Since the classical works by Student (Ref. 2) and Fisher et al. (Ref. 3), it has been known that under ‘ideal conditions’, microbial cell counts taken using a haemocytometer or colony counts on parallel plates will vary in accordance with Poisson distribution. When these conditions are fulfilled, the mean count is a direct measure of the density of the bacterial population, and such an estimate is the most accurate. ‘Any significant departure from the theoretical [Poisson] distribution is a sign that the mean may be wholly unreliable’ (Ref. 3). Modern studies show that the plate counts from, for example, the Ames Salmonella assay (Ref. 4–6) or the microbiological quality control of food and drug products (Ref. 7) too often (∼50% of experimental data sets) exhibit significant extra-Poisson variability, also called ‘over-dispersion’. This phenomenon remains a serious problem in quantitative microbiology. Biometricians try to solve it by inventing sophisticated statistical models and corresponding tests for this phenomenon. However, introducing additional parameters into such models complicates the analysis and reduces its efficacy and reliability (Ref. 5–8 and references therein). The problem is so serious that, in a recent publication (Ref. 7), German microbiologists and biometricians suggested the introduction of non-Poisson statistical models into national GLP standards for microbiological (epidemiological) control of the safety and quality of foods and drugs. One of the main reasons for all this might be the low precision of traditional plating techniques. Traditionally, two plating procedures are the most common.

Lesions with great loss of skin and extensive burns are usually treated with heterologous skin grafts, which may lead rejection. Cell therapy with mesenchymal stem cells is arising as a new proposal to accelerate the healing process. We tested a new therapy consisting of sodium carboxymethylcellulose (CMC) as a biomaterial, in combination with adipose-derived stem cells (ADSCs), to treat skin lesions in an in vivo rat model. This biomaterial did not affect membrane viability and induced a small and transient genotoxicity, only at the highest concentration tested (40 mg/mL). In a rat wound model, CMC at 10 mg/mL associated with ADSCs increased the rate of cell proliferation of the granulation tissue and epithelium thickness when compared to untreated lesions (Sham), but did not increase collagen fibers nor alter the overall speed of wound closure. Taken together, the results show that the CMC is capable to allow the growth of ADSCs and is safe for this biological application up to the concentration of 20 mg/mL. These findings suggest that CMC is a promising biomaterial to be used in cell therapy.

The organoselenium compound, dicholesteroyl diselenide (DCDS) is a structural analogue of diphenyl diselenide (DPDS) and may be considered as a promising antioxidant drug in vivo. Nevertheless, little is known about the toxicological properties of DCDS. In the present study we evaluated the cytotoxic, genotoxic and mutagenic properties of DCDS in Chinese hamster lung fibroblasts (V79) and in strains of the yeast Saccharomyces cerevisiae, proficient and deficient in several DNA-repair pathways. The results with V79 cells show that DCDS induced cytotoxicity, GSH depletion and elevation of lipid peroxidation at lower concentrations than did DPDS. DCDS also generated single- and double-strand DNA breaks in V79 cells, both in the presence and in the absence of metabolic activation, as revealed by alkaline and neutral comet assays. Moreover, the induction of oxidative DNA base-damage was demonstrated by means of a modified comet assay with formamidopyrimidine-DNA glycosylase and endonuclease III. Treatment with DCDS also induced micronucleus formation in V79 cells as well as point and frame-shift mutations in a haploid wild-type strain of S. cerevisiae. Yeast mutants defective in base excision-repair proteins were the most sensitive to DCDS. Pre-incubation with N-acetylcysteine reduced DCDS's oxidative, genotoxic and mutagenic effects in yeast and in V79 cells. Our findings indicate that the presence of cholesteroyl substituents in DCDS results in elevation of its cytotoxic and genotoxic potential compared with that of DPDS in yeast and in V79 cells. However, due to dose-dependent contrasting behaviour of organoselenium compounds and differences in their toxicity in in vitro and in vivo systems, further studies are needed in order to establish the non-toxic concentration range for treatment in mammals.

Piper gaudichaudianum Kunth is used in popular medicine as anti-inflamatory and against liver disorders. One of the most studied components of the plant is the essential oil for which chemical analysis revealed (E)-nerolidol as major compound. Recently, we have shown that P. gaudichaudianum essential oil possesses strong cytotoxic effects in mammalian V79 cells. The aim of this study was to analyze the cytotoxicity and mutagenicity of P. gaudichaudianum essential oil and nerolidol using Saccharomyces cerevisiae as model study. Treatment of the XV185-14c and N123 strains with essential oil and nerolidol led to cytotoxicity but did not induce mutagenicity. Our results revealed an important role of base excision repair (BER) as the ntg1, ntg2, apn1 and apn2 mutants showed pronounced sensitivity to essential oil and nerolidol. In the absence of superoxide dismutase (in sod1Δ mutant strain) sensitivity to the essential oil and nerolidol increased indicating that this oil and nerolidol are generating reactive oxygen species (ROS). The ROS production was confirmed by DCF-DA probing assay in Sod-deficient strains. From this, we conclude that the observed cytotoxicity to P. gaudichaudianum essential oil and nerolidol is mainly related to ROS and DNA single strand breaks generated by the presence of oxidative lesions.

By isolating putative binding partners through the two-hybrid system (THS) we further extended the characterization of the specific interstrand cross-link (ICL) repair gene PSO2 of Saccharomyces cerevisiae. Nine fusion protein products were isolated for Pso2p using THS, among them the Sak1 kinase, which interacted with the C-terminal β-CASP domain of Pso2p. Comparison of mutagen-sensitivity phenotypes of pso2Δ, sak1Δ and pso2Δsak1Δ disruptants revealed that SAK1 is necessary for complete WT-like repair. The epistatic interaction of both mutant alleles suggests that Sak1p and Pso2p act in the same pathway of controlling sensitivity to DNA-damaging agents. We also observed that Pso2p is phosphorylated by Sak1 kinase in vitro and co-immunoprecipitates with Sak1p after 8-MOP+UVA treatment. Survival data after treatment of pso2Δ, yku70Δ and yku70Δpso2Δ with nitrogen mustard, PSO2 and SAK1 with YKU70 or DNL4 single-, double- and triple mutants with 8-MOP+UVA indicated that ICL repair is independent of YKu70p and DNL4p in S. cerevisiae. Furthermore, a non-epistatic interaction was observed between MRE11, PSO2 and SAK1 genes after ICL induction, indicating that their encoded proteins act on the same substrate, but in distinct repair pathways. In contrast, an epistatic interaction was observed for PSO2 and RAD52, PSO2 and RAD50, PSO2 and XRS2 genes in 8-MOP+UVA treated exponentially growing cells.

The objective of present study was to evaluate the antioxidant and anticonvulsant activities of dichloromethane fraction (DMF) from Platonia insignis Mart., Clusiaceae. The DMF from P. insignis (2 mg/kg) was tested by intraperitoneal (i.p.) to evaluate effects on lipid peroxidation level, nitrite formation, as well as on locomotor and anticonvulsant activities. Wistar rats were treated with, (saline/Tween 80 0.5%, i.p., control group), DMF (2 mg/kg, i.p., DMF group), pilocarpine (400 mg/kg, i.p., P400 group), or the combination of DMF (2 mg/kg, i.p.) and pilocarpine (400 mg/kg, i.p., DMF plus P400). After the treatments all groups were observed for 24 h. In P400 group rats there was a decrease in the motor activity when compared with control group. In DMF plus P400 co-administered rats was observed an increase in motor activity when compared with P400 group. In P400 group rats there was a significant increase in lipid peroxidation and nitrite levels. In DMF plus P400 co-administered rats, antioxidant treatment significantly reduced the lipid peroxidation level and nitrite content after seizures. Previous findings strongly support the hypothesis that oxidative stress occurs in rat striatum during pilocarpine-induced seizures, and our results imply that strong neuprotective effect on this brain region could be achieved using DMF from P. insignis.

Diphenyl diselenide (DPDS) is an electrophilic reagent used in the synthesis of a variety of pharmacologically active organoselenium compounds. Studies have shown its interesting pharmacodinamic properties, as antioxidant, antimutagenic and antitumoral effects. Here we report the antigenotoxic properties of DPDS against tamoxifen (TAM)-induced oxidative DNA damage in MCF-7 cultured cell line. We determined the cytotoxicity by lactate dehydrogenase (LDH) leakage assay and evaluated oxidative DNA damage by modified comet assay employing the enzymes formamidopyrimidine DNA-glycosylase (Fpg) and endonuclease III (Endo III). Our results demonstrate that the cellular effects of DPDS appear to be complex and concentration-dependent. The present findings show that DPDS is not genotoxic (at concentrations lower than 2.0μmol/L) in MCF-7 cells, as observed in the modified comet assay. Moreover, DPDS protects against TAM-induced oxidative DNA damage, probably by its antioxidant activity, without interfering with its cytotoxicity. In this manner, the treatment with low concentrations of DPDS, a synthetic organoselenium compound, could be used as a potent antigenotoxic agent to prevent the risk of cancer induction triggered by tamoxifen hormone therapy. Thereby, more studies concerning the toxicity of DPDS and its structural derivatives are still necessary for future safe therapeutic application and development of novel chemopreventive compounds for combined therapy in breast cancer.

Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.

Patulin, a known mycotoxin, is considered a significant contaminant in apples, apple-derived products and feeds. This study investigated the genotoxic effects of patulin in multiple organs (brain, kidney, liver and urinary bladder) of mice using an in vivo comet assay. We assessed the mechanism underlying this genotoxicity by measuring the GSH content and the thiobarbituric acid-reactive species (TBARS) level. Male CF-1 mice were given 1.0–3.75 mg/kg patulin intraperitoneally. The effect of patulin was dose-dependent and the highest patulin dose induced DNA strand breaks in the brain (damage index, DI, in hippocampus increased from 36.2 in control animals to 127.5), liver (44.3–138.4) and kidneys (31.5–99); decreased levels of GSH (hippocampus – from 46.9 to 18.4 nmol/mg protein); and an increase in lipid peroxidation (hippocampus – from 5.8 to 20.3 MDA equivalents/mg protein). This finding establishes an interrelationship between the pro-oxidant and genotoxic effects of patulin. Pre-treatment administration of N-acetyl-cysteine reduced patulin-induced DNA damage (hippocampus – DI from 127.5 to 39.8) and lipid peroxidation (hippocampus – 20.3 to 12.8 MDA equivalents/mg protein) by restoring cellular GSH levels, reinforcing the positive relationship between patulin-induced GSH depletion and DNA damage caused by systemic administration of this mycotoxin.

Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.